The onset of DNA replication in mammalian cells is known to be regulated by cytoplasmic factors that act as positive regulatory signals. Acid-soluble nucleotides are likely to be used as intracellular signals because of their metabolic lability allowing their intracellular levels to fluctuate rapidly in response to conditions which affect growth. The objectives of this project include the understanding of how nucleotides act in the regulation of DNA replication. Diadenosine 5', 5'''-P1,P4-tetraphosphate (Ap4A), a nucleotide formed in the back reaction of the first step of protein synthesis, was shown to be related to the proliferative activities of mammalian cells and to bind to DNA polymerase alpha with high substrate specificity. Recently, we have demonstrated that the nuclear compartment pools of ATP and ADP can act as S-phase regulators. DNA replication in isolated nuclei in vitro was inhibited by high ATP levels and high ATP/ADP ratios. Studies of intact cells in vivo were consistent with the in vitro results: although the total cellular pools of ATP and ATP/ADP ratios increase upon progression of mammalian cells through the G1 and into the S phase of their cycle, the nuclear compartment pools of adenine nucleotides and nuclear ATP/ADP ratios decrease upon entry into S phase of the cell cycle. Moreover, two nuclear enzymatic activities, nuclear ATPase and AMP deaminase, were shown to increase upon entry of mammalian cells into the S phase of their cycle and may be responsible for the cell cycle-related fluctuations in the nuclear pool sizes of adenine nucleotides. We will conduct experiments designed to establish the regulatory role and site of action of Ap4A and ATP in relation to DNA replication. We plan to study the allosteric effects of Ap4A, ATP and ADP on enzymatic activities that are associated with proteins of the replication complex. Studies of regulatory mechanisms will be conducted on synchronized, intact and permeabilized cells as well as on isolated nuclei. Cell lines in which activation of host cell DNA replication is achieved by infection with tumor viruses also will be utilized.